The AMP-activated protein kinase (AMPK), a mammalian member of the AMPK/SNF1 protein kinase family, is a central regulator of many aspects of metabolism, including fatty acid and cholesterol synthesis, fatty acid oxidation, lipolysis and glucose homeostasis and of cardiac rhythmicity, ion channel function and apoptosis. The regulation of acetyl-coA carboxylase (ACC) by AMPK controls not only fatty acid metabolism, but also fuel utilization in muscle and possibly functions of the beta cell and of central appetite control. As such, this work taken together will contribute to expanded knowledge of the pathophysiology and therapy of diabetes mellitus, hyperlipidemia and obesity. Three Specific Aims are proposed which focus on how knowledge of the structure and regulation of AMPK can contribute to knowledge of its function, including the regulation of ACC. First, we propose a comprehensive study of the serial alterations in AMPK alpha and beta subunit phosphorylation, cellular trafficking and AMPKK activity during the activation or inactivation of AMPK activity in response to cellular metabolic stress. We plan to investigate the structural determinants of the differential subcellular localization of the AMPK alpha catalytic subunits in order to better understand the roles and regulation of each in the intact cell. Second, we propose to undertake a comprehensive investigation of the functions of normal and mutated gamma subunits (gamma1, gamma2, gamma3) in cultured cell systems to extend knowledge of the roles of the gamma subunit on AMPK heterotrimer assembly, activity, localization and targeting. As part of these studies, we will use the regulation of ACCalpha, ACCbeta and fatty acid oxidation in selected cell systems to "test" the fidelity of observed in vitro changes in AMPK activity to an important intracellular target and to provide needed information about the role of AMPK regulation of these two ACC isoenzymes in the regulation of fatty acid oxidation. Third, we plan to exploit our observation of the ability of mutated gamma subunits to alter cellular AMPK activity in cultured cells in the study of a whole animal model, namely the transgenic expression of R70Qgamma1 in mouse skeletal muscle. These studies, developed as a paradigm by which to regulate AMPK activity selectively in tissues via expression of mutated gamma subunits, will focus on the impact of expression of this transgene on AMPK activity, heterotrimer composition, downstream targeting of ACC and fatty acid oxidation, muscle exercise performance and glucose transport. Our overall emphasis, building knowledge of the structure/function of the complex AMPK enzyme, is necessary to understand its important roles in cellular metabolic regulation.